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Pyruvate kinase
Pyruvate kinase, E.C. number 2.7.1.40 (1), is a transferase that catalyzes the final step of the glycolytic pathway. Pyruvate kinase catalyzes the dephosphorylation of phosphonolpyruvate (PEP) to form pyruvate and energize ADP to ATP. Enzyme Structure In mammals, four pyruvate kinase isoenzymes exist. The predominant forms found in hepatocytes and erythrocytes are encoded by the PKLR gene, while the PKM gene encodes PKM1 and PKM2. PKM1 is the primary isoenzyme found in muscle and brain cells and PKM2, formed from alternative exon splicing of PKM, is present in various tissues such as lung, pancreas, embryonic, and tumor cells (2). PKM2 is also present in significant quantities in the brain (2). The human M1 isoenzyme of pyruvate kinase exists as a homotetramer with high affinity for PEP, while PKM2 exists as a low-affinity dimer and forms a high-affinity tetramer only when allosterically modified by fructose-1,6-biphosphate (3). Each subunit of PKM1 consists of 531 amino acids and weighs 58 062 daltons (4, 5). Each subunit of the mammalian pyruvate kinase consists of four domains: a classic barrel consisting of β strands and α helices; a smaller, irregular barrel; the C-terminal domain consisting of β strands and α helices; and an N-terminal helix-turn-helix motif (6). The active site of human PKM1 and PKM2 consists of lysine 270 and residues 363-368, and the allosteric fructose 1,6-biphosphate binding site may involve lysine 433, serine 434, serine 437, and glycine 520; or arginine 489, glycine 518, and tryptophan 482; or phenylalanine 521 and glycine 514 (7). Normal Function Pyruvate kinase catalyzes the final step of the glycolytic pathway, in which a phosphate group from PEP is removed to form pyruvate and said phosphate group is transferred to ADP to form ATP. This reaction requires the presence of the cofactors K+ and Mg2+. The complete reaction mechanism involves a nucleophilic attack on the phosphorus atom of PEP by the β-phosphoryl oxygen of ADP. The phosphate group is transferred from PEP to ADP and results in the formation of ATP. The loss of its phosphate group results in the conversion of PEP to the intermediate enolpyruvate. Enolpyruvate then tautomerizes to pyruvate with the addition of a hydrogen ion. K+ and Mg2+ stabilize the negative charge of the oxygen atoms during the reaction (9). This reaction is allosterically inhibited through negative feedback from the products, ATP and pyruvate (6). Observations in Alzheimer's Disease The activity of pyruate kinase in the brain cells of Alzheimer’s disease patients has been assessed infrequently in the literature. In a study by Iwangoff et al., an in vitro assay was conducted (10). This experiment measured the activity of pyruvate kinase from the autopic temporal lobes of subjects in assays with known concentrations of pyruvate kinase as well as optimized substrate, cofactor, and activator concentrations and physiological conditions. Additionally, the assay contained an excess of lactate dehydrogenase and a set concentratin of NADH. In the assay, upon the formation of pyruvate by pyruvate kinase, lactate dehydrogenase converts pyruvate to lactate and NADH to NAD+. The concentration of NADH in solution can be measured through absorption; NADH is the only reactant in the mixture that has a notable extinction coefficient at 340nm. The activity of pyruvate kinase is thus assessed by the change in absorption. This study found no change in pyruvate kinase activity in the autopic temporal lobes of Alzheimer’s disease patients relative to healthy controls (Figure 3) (10). A similar protocol was carried out by Bigl et al. to measure pyruvate kinase activity using updated conditions (Figure 4) (11). The improved protocol confirmed Iwangoff et al.’s findings of no significant change in cerebral cortex pyruvate kinase activity (11). Bigl et al. found a significant increase in pyruvate kinase activity in the frontal cortices of Alzheimer’s disease patients and found no significant change in pyruvate kinase activity of the basal forebrain, hippocampus, striatum, and occipital cortex (11). The increase of pyruvate kinase activity in the frontal cortex may be accounted for by the increase of astrocytes in this region of the brain (11). Although the neuronal degradation associated with Alzheimer’s disease likely decreases neuronal glycolysis, the activity of pyruvate kinase throughout the brain remains relatively constant due to the increase of astrocytes (11). The findings of pyruvate kinase activity support the explanation that additional pyruvate is formed in astrocytes to allow the transfer of energy to neurons in the form of lactate, following the astrocyte-neuron lactate shuttle model (12). ---- Back to homepage. Works Cited 1. RCSB Protein Data Bank. Structure of M2 pyruvate kinase in complex with phenylalanine. http://www.rcsb.org/pdb/explore/explore.do?structureId=4FXF (accessed November 21, 2013) 2. Tolle, S.W., Dyson, R.D., Newburgh, R.W. and Cardenas, J.M. (1976) PYRUVATE KINASE ISOZYMES IN NEURONS, GLIA, NEUROBLASTOMA, AND GLIOBLASTOMA.J.Neurochem. 27, 1355-1360 3. Cortes-Cros, M., Hemmerlin, C., Ferretti, S., Zhang, J., Gounarides, J.S., Yin, H., Muller, A., Haberkorn, A., Chene, P., Sellers, W.R. and Hofmann, F. (2013) M2 isoform of pyruvate kinase is dispensable for tumor maintenance and growth. Proc.Natl.Acad.Sci.U.S.A. United States 110, 489-494 4. PDB ID: 3SRF. Morgan, H.P., O'Reilly, F., Palmer, R., McNae, I.W., Nowicki, M.W., Wear, M.A., Fothergill-Gilmore, L.A., Walkinshaw, M.D. Allosetric regulation of M2 pyruvate kinase from Homo sapiens. 5. Uniprot entry name KPYM_HUMAN. The UniProt Consortium. Update on activities at the Universal Protein Resource (UniProt) in 2013. Nucleic Acids Res. 41: D43-D47 (2013). 6. Mattevi, A., Bolognesi, M., and Valentini, G. (1996) The allosteric regulation of pyruvate kinase. FEBS Lett. 389, 15-19 7. Eigenbrodt, E.; Mazurek, S. Pyruvate kinase type M2: amino acid sequence. http://www.metabolic-database.com/html/m2-pk_amino_acid_sequence.html (accessed November 21, 2013). 8. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC. 9. Dann, L. G., and Britton, H. G. (1978) Kinetics and mechanism of action of muscle pyruvate kinase. Biochem.J. 169, 39-54 10. Iwangoff, P., Armbruster, R., Enz, A., & Meier-Ruge, W. (1980). Glycolytic enzymes from human autoptic brain cortex: normal aged and demented cases. Mechanisms of ageing and development, 14(1-2), 203–209. 11. Bigl, M.; Bruckner, M. K.; Arendt, T.; Bigl, V.; Eschrich, K. Activities of key glycolytic enzymes in the brains of patients with Alzheimer's disease. J Neural Transm 1999, 106, 499-511. 13. Newington, JT. Harris, RA. Cumming, RC. (2013) Reevaluating Metabolism in Alzheimer's Disease from the Perspective of the Astrocyte-Neuron Lactate Shuttle Model. Journal of Neurodegenerative Diseases. 2013, 1-13